Cytokines are important molecules produced by cells of the immune system. Cellular responses to cytokines are regulated by cell surface receptors, polypeptide chains that individually and/or coordinately bind cytokines and transmit signals that regulate activation of cellular genes. The affinities of these receptors regulate the magnitude of the cellular response, which can impact the pattern and determine the physiologic outcome. Regulation of cytokine responses can therefore determine if a cell is activated, divides, differentiates, becomes tolerant or dies. Regulation of cytokine response can also determine if auto-immune cells are permitted to survive and proliferate.
Each cytokine receptor contains one or more cytokine-specific polypeptide chains. As discussed below, when cytokines bind to their cognate receptors, signals through these multiple cell surface polypeptide chains of cytokine receptors control cell growth, activation and differentiation, and appear to be critical in regulating the generation and maintenance of immune responses (Sugamura et al., Adv. Immunol. 59:225-277 (1995)).
Briefly, immune responses are initiated by antigen presenting cells (APC) which display peptide fragments of processed foreign antigen in association with MHC class II molecules on their surfaces to CD4+helper T lymphocytes (T helper cells or Th cells) which interact with these APC's. The Th cells are activated when they recognize particular epitopes of a foreign antigen displayed on the appropriate APC surface for which the Th cells express a specific T cell receptor (TcR). In addition to the TcR interaction with a peptide/MHC complex, T cells require a second cognate costimulatory signal, usually through a cell surface receptor, for eg. CD28 (Harding, Nature 356:607-609 (1992) interacting with ligands for CD28 expressed on APC including B7-1 and B7-2 (Bluestone, Immunity 2:555-559 (1995). Productive engagement of these two types of cell surface receptors primes the T cells to make and to respond to one or more cytokines secreted or released by activated cells. A key cytokine is interleukin-2 (IL-2), which stimulates and supports cell division, increasing the number of interacting cell types and hence the magnitude of the immune response.
In addition to regulating the magnitude of immune responses, the pattern of cytokines released at the onset of an immune challenge affects the subsequent choice of which immune effector pathways are activated (Paul and Seder, Cell 76:241-251 (1993). These cytokines are released by several cell types involved in initiating the immune response, including APC and CD4+helper Th cells which interact with the APCs. Activated Th cells secrete cytokines which, together with the APC-derived cytokines, direct their differentiation into one of several types of Th cells. These different types of Th cells are then responsible for activating diverse effector mechanisms including killer T cell activation, B cell antibody production and macrophage activation. The choice between effector mechanisms is mediated largely by which cytokines are produced by the activated Th cells.
Th cells can be divided into three subgroups based on their cytokine secretion patterns (Fitch et al., Ann. Rev. Immunol., 11, pp. 29-48 (1993); Mosmann and Sad, Immunol. Today 17:138-146 (1996)). These subgroups are called Th0, Th1 and Th2. In humans, the Th1 pattern of cytokine secretion has been generally associated with cellular immunity and resistance to infection with viruses and intracellular parasites. The Th1 cytokines such as IFN .gamma. and IL-2 tend to activate macrophages, natural killer cells and cytotoxic T cells. Other cytokines produced by APCs, such as IL-7 and IL-15, may also participate in the activation of these cytotoxic functions. In addition to their protective functions, Th1 cytokines promote deleterious inflammatory responses such as delayed type hypersensitivity. Pathological Th1 responses are also associated with a number of organ-specific and systemic autoimmune conditions and with chronic inflammatory diseases, and play an important role in cellular rejection of tissue grafts and organ transplants. In contrast, the Th2 pattern of cytokine secretion (IL-4, IL-5, IL-6, IL-9 and IL-10) promotes the full expansion and maturation of B cells thereby providing humoral protection, for example, against extracellular pathogens (Howard et al., "T cell-derived cytokines and their receptor", Fundamental Immunology, 3d ed., Raven Press, New York (1993)). Th2 cytokines such as IL-4 and IL-9 also increase eosinophil and mast cell production. But like Th1 responses, deleterious Th2 responses can lead to pathologic conditions, including IgE antibodies associated with allergic responses, autoimmune antibodies such as those in idiopathic thrombocytopenia, myasthenia gravis, and systemic lupus erythematosus, and anti-graft antibodies.
The pattern and magnitude of cytokine responses can also be used to negatively regulate activated cells. The absence of appropriate cytokine dependent signaling to activated cells (physiologically, an inadequate or temporally discordant signal) usually results in their death. In some circumstances it can lead to a so called tolerant state, also called unresponsiveness or anergy, where cells survive but fail to respond to subsequent stimuli (Schwartz, Science, 248, 1349-1356 (1990); Jenkins et al., J. Immunol., 140, 3324-3330 (1988). Thus, inhibition of cytokine signaling to activated cells may represent a physiological means of regulating for eg. autoreactive cells. It also may be therapeutically useful in treating autoimmune and inflammatory diseases.
One way to block cytokine responses is to target the receptor. In the case of IL-2, the receptor is composed of three distinct polypeptide chains: alpha, beta and .gamma. common (hereinafter "gc"). The alpha chain specifically binds to IL-2 but has no capacity to signal the cell which expresses it. The beta chain binds IL-2 poorly (K.sub.D =1 micromolar), but with the alpha chain creates a two chain receptor with an affinity that exceeds that of either chain alone. The gc chain binds IL-2 very weakly, if at all, but combines with the alpha and beta chains to create a three chain receptor with very high affinity [10 picomolar] for IL-2 (Sugamura et al., Adv. Immunol. 59:225-277 (1995)). Other combinations of the IL-2 receptor chains are also possible. For example, on NK cells the beta and gc chains combine to form an IL-2 receptor of intermediate affinity (K.sub.D =1 nanomolar).
The gc chain is a cell surface polypeptide component of cytokine receptors and forms part of the receptors for several other interleukins besides IL-2 such as IL- 4, 7, 9, and 15. The extracellular region of the gc chain of these IL receptors (IL-R) is composed of 2 FNIII type domains, each comprised of 7 strands connected by loop sequences which are presumed to form intermolecular contacts (see structural model derived for the IL-4/IL-4 chain gc chain complex, Gustchina et al., Proteins: Structure, Function and Genetics 21:140 (1995)), and for the IL-2/IL-2R complex, Bamborough et al., Structure 2:839-851 (1994)).
In the case of the IL-4 receptor, the gc chain is paired only with a cytokine binding alpha chain; no additional beta chain has been identified to date. The same is true for the IL-7 and IL-9 receptors; each brings its own cytokine binding alpha chain to combine with the gc chain to create a complete cooperating two chain receptor. The receptor for IL-15 is composed of an alpha chain specific for IL-15 together with the beta chain of the IL-2 receptor and the gc chain. The participation of two and sometimes three polypeptide chains in the functional receptor thus adds another layer of complexity to the regulation of the immune response. Since the gc chain is shared by a number cytokine receptors, blocking the function of the gc chain may affect cells that depend on signaling from IL-2, IL-4, IL-7, IL-9 or IL-15.
Several mAbs which block the function of the murine and human gc chains have been described in the literature. Sugamura and colleagues developed a mAb which binds to murine gc chain and which blocks responses to IL-4, IL-7 and IL-9. The mAb needed auxiliary molecules to inhibit IL-2 (i.e. the mAb failed to inhibit IL-2 responses when used alone). See Kondo et al., Science, 262, pp. 1874-1877 (1993); Kondo et al., Science, 263, pp. 1453-1454; Kimura et al., International Immunology, 7, pp. 115-120 (1994). See also U.S. Pat. No. 5,582,826 (anti-human gc antibody significantly inhibiting cellular IL-2 activity requires an auxiliary antibody).
He et al. (J. Immunol., 154, pp. 1596-1605 (1995) reported two mAbs which bind to murine gc chain. When used without auxiliary molecules (i.e., when used alone) one mAb partially blocked IL-4 but failed to block IL-2 or IL-7, and the other mAb partially blocked IL-7 but failed to block IL-4 or IL-2. These results demonstrate that the gc chain is employed in distinct ways by different receptors. This observation indicates that screening for mAbs that bind to gc chain or that block the cellular response to a given cytokine of the group including IL-2, IL-4, IL-7 IL-9 and IL-15, will not necessarily produce mAbs that can block the cellular responses to any of the other cytokines of this group.
Moreover, given the extremely high affinity (10 pM) of this group of cytokine receptors for their respective cytokines, it would be difficult to achieve effective inhibition using a blocking agent of much lower affinity (e.g., 1-100 nanomolar affinity typical of a mAb) that is competing with cytokine for binding to the same receptor, particularly in the presence of high concentrations of cytokine.
There are no mAbs reported in the prior art that bind to gc chain and block cellular responses to IL-2 when used in the absence of auxiliary molecules. Nor has any single mAb been reported which can block cellular responses to any subset of cytokine which includes IL-2. Indeed, all mAb specific for gc chain developed to date require the addition of an auxiliary molecules i.e., a second compound which could act in concert with that agent to inhibit the cellular response to IL-2.
Monoclonal antibodies (mAbs) that block the function of the gc chain and thereby block cellular responses to cytokines which employ the gc chain in their receptors could provide useful agents to treat various Th cell-based immunological conditions. To date, however, such treatment generally has employed immunomodulatory and immunosuppressive agents as well as a number of drugs (eg. gold or penicillamine) with poorly characterized mechanisms. Three general immunosuppressive agents used currently are steroids, cyclosporine and azathioprine. These non-specific agents are generally required chronically and in high doses are associated with significant toxicity, particularly nephrotoxicity and hepatotoxicity, as well as other adverse side effects.
Useful agents of this kind would include those which block cellular responses to any one of the group of cytokines that have the gc chain in their receptors, to selected members of these, or to all members of this group.